Engraftment Of Hepatocytes Research Articles

Long Term Liver Engraftment of Functional Hepatocytes Obtained from Germline Cell-Derived Pluripotent Stem Cells.

One of the major hurdles in liver gene and cell therapy is availability of ex vivo-expanded hepatocytes. Pluripotent stem cells are an attractive alternative. Here, we show that hepatocyte precursors can be isolated from male germline cell-derived pluripotent stem cells (GPSCs) using the hepatoblast marker, Liv2, and induced to differentiate into hepatocytes in vitro. These cells expressed hepatic-specific genes and were functional as demonstrated by their ability to secrete albumin and produce urea. When transplanted in the liver parenchyma of partially hepatectomised mice, Liv2-sorted cells showed regional and heterogeneous engraftment in the injected lobe. Moreover, approximately 50% of Y chromosome-positive, GPSC-derived cells were found in the female livers, in the region of engraftment, even one month after cell injection. This is the first study showing that Liv2-sorted GPSCs-derived hepatocytes can undergo long lasting engraftment in the mouse liver. Thus, GPSCs might offer promise for regenerative medicine.

Experimental hepatocyte xenotransplantation--a comprehensive review of the literature.

Hepatocyte transplantation (Tx) is a potential therapy for certain diseases of the liver, including hepatic failure. However, there is a limited supply of human livers as a source of cells and, after isolation, human hepatocytes can be difficult to expand in culture, limiting the number available for Tx. Hepatocytes from other species, for example, the pig, have therefore emerged as a potential alternative source. We searched the literature through the end of 2014 to assess the current status of experimental research into hepatocyte xenoTx. The literature search identified 51 reports of in vivo cross-species Tx of hepatocytes in a variety of experimental models. Most studies investigated the Tx of human (n = 23) or pig (n = 19) hepatocytes. No studies explored hepatocytes from genetically engineered pigs. The spleen was the most common site of Tx (n = 23), followed by the liver (through the portal vein [n = 6]) and peritoneal cavity (n = 19). In 47 studies (92%), there was evidence of hepatocyte engraftment and function across a species barrier. The data provided by this literature search strengthen the hypothesis that xenoTx of hepatocytes is feasible and potentially successful as a clinical therapy for certain liver diseases, including hepatic failure. By excluding vascular structures, hepatocytes isolated from genetically engineered pig livers may address some of the immunological problems of xenoTx.

275. Pancreatic Transdiffereniation of Human Hepatocytes in the Livers of a Humanized Mouse Model

Type I diabetes (TID) results from the T-cell mediated autoimmune destruction of the insulin producing pancreatic beta (β) cells. Exogenous insulin therapy cannot achieve precise physiologic control of blood glucose concentrations and debilitating complications develop, which greatly increase morbidity and mortality for patients. Gene therapy is one strategy that holds considerable promise to cure TID. In earlier studies using an improved surgical technique that isolates the liver from the circulation, we introduced furin-cleavable human insulin (INS-FUR) to the livers in several animal models of diabetes. Reversal of diabetes with normal glucose tolerance and pancreatic transdifferentiation of the liver tissue was seen in streptozotocin (STZ)-diabetic Wistar rats, non-obese spontaneous diabetic mice and pancreatecomised diabetic pigs using the HMD lentiviral vector to deliver the INS-FUR. The aims of the present study were (1) to determine if STZ-diabetes could be reversed in the FRG mouse model in which chimeric mouse-human livers can be readily established, and (2) to determine if pancreatic transdiffereniation was evident in the engrafted human hepatocytes. Successful engraftment of human hepatocytes was verified by measurement of human albumin levels and diabetes was subsequently induced by STZ. Following delivery of the INS-FUR vector (3.5×108transduction units) to overtly diabetic FRG mice (blood glucose >13mM), mice were monitored for changes in body weight and blood glucose levels. Reversal of diabetes was seen for a period of 2 months, which was the experimental endpoint. Intraperitoneal glucose tolerance tests (IPGTTs) of the INS-FUR-transduced animals were not significantly different from nondiabetic, control animals. RT-PCR showed expression of human insulin, glucagon and somatostatin and a number of human β-cell transcription factors, including Pdx1, Neurod1 and Neurogenin 3, indicating pancreatic transdifferentiation of hepatocytes in vivo. Double fluorescent staining for human albumin and insulin was seen in transduced liver samples. We have shown, for the first time that using our technology, human hepatocytes can be induced to undergo pancreatic transdiffereniation in vivo. These human hepatocytes have likely contributed to the reversal of diabetes seen in the animals. Our data suggest that this regimen may ultimately be employed clinically to cure TID.

705. Selective Advantage of Hepatocytes Expressing Wild-Type Alpha-1 Antitrypsin (AAT) in a Novel Human Liver Xenograft Model: A Model for Correction of a Gain-Of-Function Mutation

Hepatocytes play a central role in energy metabolism and secretion of plasma proteins and represent an important target for gene therapy of single gene disorders. Alpha-1 antitrypsin (AAT) deficiency is one such disorder, in which one common missense mutation (E342K, known as the PI*Z allele) results in impaired secretion of AAT. In the majority of patients with AAT deficiency, this causes a lung disease due to a lack of the normal AAT-mediated protection of lung elastin from neutrophil elastase. In a subset of patients, accumulation of Z-mutant AAT protein triggers hepatocyte injury leading to inflammation and cirrhosis. As vector-mediated methods for correction of these two defects have been developed for both in vivo and ex vivo use, we sought to determine whether correction of the Z-mutant defect in hepatocytes might confer a selective advantage for repopulation of hepatocytes within an intact liver. To test this concept, a transgenic mouse strain expressing a human PI*Z allele was crossed with the NOD-SCID-gamma chain knockout (NSG) strain to create a recipient strain (PI*Z-NSG) for human hepatocyte xenotransplantation. Initial comparisons indicated that PI*Z-NSG recipients support more efficient engraftment of normal (wt-AAT) human primary hepatocytes as compared with NSG recipients. A number of procedures to optimize primary hepatocyte engraftment, and pretreatment of the recipient with monocrotaline further accentuated the level of engraftment. Finally, we propose to test the paradigm of using the selective advantage of wt-AAT hepatocytes to model gene and cell therapy of AAT deficiency, through the introduction of a dual-function lentivirus vector expressing both a myc-tagged wild-type AAT and a synthetic miRNA to knockdown the endogenous allele. The NSG-PI*Z recipient strain could be used as a platform for future studies of both cell-based and genetic therapies of AAT deficiency and further could predict a selective advantage of corrected cells in AAT deficient patients. In addition this new liver xenograft model provides a setting in which to easily a reproducibly repopulate a mouse liver with human hepatocytes.

Transplantation speed offers early hepatocyte engraftment in acute liver injured rats: A translational study with clinical implications.

The impact of the rate of intraportal hepatocyte transplantation on early engraftment and repopulation is unclear. The aim of this study was to address this and to improve the engraftment and repopulation efficiencies of hepatocyte transplantation for the treatment of a rat model of acute liver failure in a clinically useful way without preconditioning. Acute hepatic injury was induced into Sprague-Dawley rats with D-galactosamine. Hepatocytes were infused intraportally over a period of 30, 70, or 100 seconds to study early engraftment (2 days) and repopulation (7 days). Three groups had significant differences in hepatocyte engraftment (P = 0.018) and repopulation efficiencies (P = 0.037), and an infusion over a period of 70 seconds produced superior outcomes. After the 70-second infusion, the transplanted cells immediately transmigrated the sinusoidal endothelial layer and rarely accumulated in the portal venules, with liver function improving significantly. The mean first peak pressures, without significant differences, were 14.8 ± 6.5, 17.7 ± 3.7, and 13.6 ± 3.0 mm Hg in the 30-, 70-, and 100-second groups, respectively. Differential hepatocyte transfusion rates contributed to accelerated early engraftment and repopulation in rats with acute liver injury. These proof-of-concept findings are of clinical significance because they are easy to translate into practice.

A novel mouse model for stable engraftment of a human immune system and human hepatocytes.

Hepatic infections by hepatitis B virus (HBV), hepatitis C virus (HCV) and Plasmodium parasites leading to acute or chronic diseases constitute a global health challenge. The species tropism of these hepatotropic pathogens is restricted to chimpanzees and humans, thus model systems to study their pathological mechanisms are severely limited. Although these pathogens infect hepatocytes, disease pathology is intimately related to the degree and quality of the immune response. As a first step to decipher the immune response to infected hepatocytes, we developed an animal model harboring both a human immune system (HIS) and human hepatocytes (HUHEP) in BALB/c Rag2-/- IL-2Rγc-/- NOD.sirpa uPAtg/tg mice. The extent and kinetics of human hepatocyte engraftment were similar between HUHEP and HIS-HUHEP mice. Transplanted human hepatocytes were polarized and mature in vivo, resulting in 20–50% liver chimerism in these models. Human myeloid and lymphoid cell lineages developed at similar frequencies in HIS and HIS-HUHEP mice, and splenic and hepatic compartments were humanized with mature B cells, NK cells and naïve T cells, as well as monocytes and dendritic cells. Taken together, these results demonstrate that HIS-HUHEP mice can be stably (> 5 months) and robustly engrafted with a humanized immune system and chimeric human liver. This novel HIS-HUHEP model provides a platform to investigate human immune responses against hepatotropic pathogens and to test novel drug strategies or vaccine candidates.

Ultrasonic Histotripsy for Tissue Therapy

Hepatocyte transplantation has been considered and investigated as a promising and alternative method to liver transplantation for treating liver-based metabolic disorder in newborns over the past two decades. Although some clinical trials have been conducted and shown clinical benefits and outcomes, it is difficult to deliver and achieve a desired level of integration and transplantation of hepatocytes in the liver parenchyma. To overcome this problem, this work introduces an alternative method to a portal-infused-hepatocyte cell transplantation. To improve the level of engraftment of transplantable hepatocytes, these are injected directly into cavities generated by ultrasonic histotripsy. Histotripsy is an extracorporeal noninvasive technique which has been recently developed using high intensity focused ultrasound (HIFU) for inducing tissue fractionation with no coagulative necrosis. The exact mechanisms for the tissue fractionation are not well understood yet; but the possible mechanisms are thought to be a combination of nonlinear wave propagation effect, explosive bubble growth and ultrasonic atomization. The main objectives of this work are to demonstrate the feasibility of this new cell therapy and evaluate and distinguish between the different types of cavitation activity for either a thermally or a mechanically induced lesion. In the present work, numerical studies on the bubble dynamics (the Gilmore-Akulichev bubble model coupled with the Khokhlov-Zabolotskaya-Kuznetsov equation) and both ex- and in vivo liver experiments are conducted with histological analysis (haematoxylin and eosin stain). The numerical and the experimental results suggest that (a) the acoustic emissions emitted during the thermal ablation and the histotripsy exposure can be distinguished both numerically and experimentally and (b) the proposed cell therapy may potentially form an effective and safe clinical treatment for replacing and correcting disordered hepatocytes, although the transplantation was not conducted in this work.

Improvement of hepatocyte transplantation efficiency in the mdr2-/- mouse model by glyceryl trinitrate.

Hepatocyte transplantation could be an alternative to liver transplantation for the treatment of metabolic diseases. However, rodent models have shown that engraftment of transplanted cells in the liver is low and requires deposition of cells in hepatic sinusoids. Splanchnic vasodilatators improved hepatocyte engraftment in a rat model. Therefore, we investigated the effect of glyceryl trinitrate (GTN) on the efficacy of cell engraftment and on liver repopulation in the mdr2-knockout mouse, a model for progressive familial intrahepatic cholestasis type 3. Congenic normal mdr2 (+/+) hepatocytes were isolated by two-step collagenase perfusion and transplanted into mdr2(-/-) mice livers through the portal vein in the presence or absence of GTN. Liver repopulation was assessed by immunohistochemistry, and transplanted hepatocyte function was assessed at different times after transplantation by measurement of biliary lipid secretion and quantification of fibrosis. The number of engrafted cells in GTN-treated mice was significantly higher than that in control mice, and transplanted hepatocytes were found in a greater number of distal sinusoids. Levels of phospholipid secretion were significantly higher than those in the control group 3 months after hepatocyte transplantation (18.3 ± 2.3 vs. 5.2 ± 3.9 nmol/min/100 g, P < 0.0001), and the ratio of phospholipids to bile salt was greater (6.8 ± 1.3 vs. 3.2 ± 1.6, P = 0.03). The percentage area of liver fibrosis was also significantly reduced in GTN-treated mice (5.7% ± 2.3% vs. 12.4% ± 2.9%, P = 0.016). The use of GTN improves hepatocyte engraftment and correction of metabolic disease in mdr2 (-/-) mice. This approach might be beneficial in hepatocyte transplantation for the treatment of patients with liver diseases.

Ectopic Liver Tissue Formation in Rats with Induced Liver Fibrosis.

IntroductionThe possible alternative approach to whole-organ transplantation is a cell-based therapy, which can also be used as a “bridge” to liver transplantation. However, morphological and functional changes in the liver of patients suffering from chronic liver fibrosis and cirrhosis restrict the effectiveness of direct cell transplantation. Therefore, extra hepatic sites for cell transplantation, including the spleen, pancreas, peritoneal cavity, and subrenal capsule, could be a useful therapeutic approach for compensation of liver functions. However, a method of transplantation of hepatocytes into ectopic sites is needed to improve hepatocyte engraftment. Previously published data has demonstrated that mouse lymph nodes can support the engraftment and proliferation of hepatocytes as ES and rescue Fah mice from lethal liver failure. Thus, the aim of the study was to evaluate the engraftment of i.p. injected allogeneic hepatocytes into extra hepatic sites in albino rats with chemically induced liver fibrosis (LF).Materials and methodsAlbino rats were randomly divided into 4 groups: (1) intact group (n = 18); (2) rats with induced LF (n = 18); (3) rats with induced LF and transplanted with hepatocytes (n = 18); (4) as a control, rats were treated with cyclosporine A only (n = 18). In order to prevent an immune response, groups 2 and 3 were subjected to immunosuppression by cyclosporine A (25 mg/kg per day). LF was induced using N-nitrosodimethylamine (NDMA), i.p., 10 mg/kg, three times a week for 4 weeks and confirmed by histological analysis of the liver samples. Hepatocytes transplantation (HT) was performed two days after NDMA exposure cessation by i.p. injection of 5×106 freshly isolated allogeneic hepatocytes. Liver function was assessed by quantifying blood biochemical parameters (ALT, AST, GGT, total protein, bilirubin, and albumin) at 1 week, 1 month, and 2 months after hepatocytes transplantation (HT). To confirm a hepatocytes’ engraftment, we conducted immunohistochemical staining against HepPar1.ResultsWe observed a 30% mortality rate among rats with LF within 1 week after NDMA exposure cessation, while 100% of animals with HT survived. ALT, AST, and GGT activities and bilirubin levels were markedly elevated in blood samples of LF rats compared to the control animals. However, HT significantly improved ALT, AST, and GGT activity as well as bilirubin levels. We also observed decreased levels of total protein and albumin in the blood serum of rats with LF, while HT normalized these parameters. At the same time, we have not detected any statistical differences of the studied parameters in the group 4, which was treated with Cyclosporine A only, compared with the intact animals. HepPar1 immunohistochemical staining of the different tissue sections demonstrated the presence of engrafted hepatocytes, mainly within enlarged Peyer’s patches (aggregated lymphoid nodules in the lowest portion of the small intestine).ConclusionThe results of our study provide evidence that HT improves animal survival and liver functions. One potential reason for these results is that ectopic hepatic mass inside the Peyer’s patches can rescue rats from liver failure.

Increased Survival despite Failure of Transplanted Human Hepatocyte Implantation into Liver Parenchyma of Nude Mice with Repeated Lethal Jo2-Induced Liver Deficiency

We recently found that rat hepatocyte transplantation was efficient (liver repopulation: 2.4%) in a sublethal nude mouse model (less than 33% mortality) of repeated liver injury generated using Jo2, a mouse-specific anti-Fas antibody, at sublethal dose of 250 µg/kg for 3 weeks. Genomic analysis of the livers revealed cell cycle blockade and an antiproliferative status of circadian genes, suggesting a selective advantage. By contrast, in the present study, freshly isolated human hepatocyte transplantation performed in the same mouse model resulted in implantation of less than 6,000 cells per liver (about 0.006% repopulation) in all animals. Genomic analysis of nude mouse livers revealed a lack of P21 upregulation, while a signature of stimulation of liver regeneration was observed, including upregulation of early response genes and upregulation of circadian genes. When we translated this sublethal model to a lethal model (65% mortality) by increasing the Jo2 repeated doses to 375 µg/kg, human hepatocyte engraftment was still very low; however, animal mortality was corrected by transplantation (only 20% mortality). Genomic findings in livers from the mice of the lethal Jo2 transplanted group were similar to those of the sublethal Jo2 transplanted group, that is, no selective advantage genomic signature and signature of mouse liver regeneration. In conclusion, transplanted human hepatocytes acted as if they modified nude mouse liver responses to Jo2 by stimulating liver regeneration, leading to an increased survival rate.

Novel management of acute or secondary biliary liver conditions using hepatically differentiated human dental pulp cells.

The current definitive treatment for acute or chronic liver condition, that is, cirrhosis, is liver transplantation from a limited number of donors, which might cause complications after donation. Hence, bone marrow stem cell transplantation has been developed, but the risk of carcinogenesis remains. We have recently developed a protocol for hepatic differentiation of CD117(+) stem cells from human exfoliated deciduous teeth (SHED). In the present study, we examine whether SHED hepatically differentiated (hd) in vitro could be used to treat acute liver injury (ALI) and secondary biliary cirrhosis. The CD117(+) cell fraction was magnetically separated from SHED and then differentiated into hepatocyte-like cells in vitro. The cells were transplanted into rats with either ALI or induced secondary biliary cirrhosis. Engraftment of human liver cells was determined immunohistochemically and by in situ hybridization. Recovery of liver function was examined by means of histochemical and serological tests. Livers of transplanted animals were strongly positive for human immunohistochemical factors, and in situ hybridization confirmed engraftment of human hepatocytes. The tests for recovery of liver function confirmed the presence of human hepatic markers in the animals' blood serum and lack of fibrosis and functional integration of transplanted human cells into livers. No evidence of malignancy was found. We show that in vitro hdSHED engraft morphologically and functionally into the livers of rats having acute injury or secondary biliary cirrhosis. SHED are readily accessible adult stem cells, capable of proliferating in large numbers before differentiating in vitro. This makes SHED an appropriate and safe stem cell source for regenerative medicine.

Antibody Mediated Rejection After Hepatocyte Transplantation Combined With Partial Hepatectomy in One of Two Patients With Crigler-Najjar Type I.

Crigler-Najjar syndrome type I (CNS) is caused by a deficiency of uridine diphosphate glucuronosyltransferase. Patients are at risk of fatal brain injury due to unconjugated hyperbilirubinemia. Treatment consists of 10 - 12h phototherapy daily. Most patients undergo liver transplantation as phototherapy becomes less effective and constitutes a significant impairment in quality of life. A 13-year-old boy and an 11-year-old girl with CNS were evaluated at our center. Both patients required 8 - 12h of phototherapy daily to maintain s-bilirubin below 450 μmol/liter. Hepatocytes were isolated under good manufacturing practices from liver tissue obtained from deceased organ donors. Fresh ABO compatible hepatocytes were infused through a portal catheter. Before infusion patients underwent liver resection of segments 2 and 3 to induce liver regeneration and proliferation of transplanted hepatocytes. Immunosuppression consisted of basiliximab, tacrolimus and steroids. The girl received 5.3 x 109 viable hepatocytes at one occasion and the boy received two infusions from different donors three months apart with 2.2 and 9 x 109 viable hepatocytes. In both patients s-bilirubin increased initially after the first procedure. Thereafter s-bilirubin decreased continuously in both patients to 50% of pre-transplant levels for more than 6 months. Bile analysis showed presence of bilirubin diglucuronides in both patients after transplantation. The boy experienced a sudden increase of serum bilirubin to pre-transplant levels 6 months after the first infusion associated with a severe scabies infection. Despite intensified phototherapy s-bilirubin did not improve. Due to the scabies infection and an incorrect assumption that the cells were rejected immunosuppression was stopped. After cessation of immunosuppressive therapy the patient presented with fever, abdominal pain and elevated transaminases followed by increasing high-level donor specific antibodies indicating viable hepatocytes at time of cessation. These studies confirm that hepatocyte transplantation can be a useful treatment for CNS. Preconditioning patients with liver resection prior to cell transplantation is safe and might improve donor hepatocyte engraftment. To our knowledge this is the first report of antibody-mediated rejection in clinical hepatocyte transplantation.

Underpinnings of cellular organ replacement therapies

Underpinnings of cellular organ replacement therapies

Ex Vivo Magnetic Resonance Imaging of Transplanted Hepatocytes in a Rat Model of Acute Liver Failure

Hepatocyte transplantation is being evaluated as an alternative to liver transplantation. However, the fate of hepatocytes after transplantation is not well defined. The aims of the study were to improve hepatocyte labeling in vitro using superparamagnetic iron oxide nanoparticles (SPIOs) and to perform in vivo experiments on tracking labeled cells by magnetic resonance imaging (MRI). Human and rat hepatocytes were labeled in vitro for 16 h with clinically approved SPIOs (12.5 µg Fe/ml) and protamine sulfate (3 µg/ml) as a transfection agent. Increased cellular iron uptake was obtained, and cell viability and function were shown not to be affected by labeling. Labeled cells (2,000/µl) could be detected on T2-weighted images in vitro using a 7T MR scanner. In a rat model of acute liver failure (ALF), female recipients received intrasplenic transplantation of 2 × 10(7) male rat hepatocytes 28-30 h after intraperitoneal injection of d-galactosamine (1.2 g/kg). There were four groups (n = 4 each): vehicle injection, injection of freshly isolated cells labeled with CM-DiI, injection of cultured cells labeled with CM-DiI, and injection of cultured cells labeled with both SPIOs and CM-DiI. Ex vivo T2*-weighted gradient-echo images at 7T MRI were acquired at day 7 post-ALF induction. Six days after transplantation, SPIOs were detected in the rat liver as a decrease in the MRI signal intensity in the surviving animals. Histologically, most of the SPIOs were located in Kupffer cells, indicating clearance of labeled hepatocytes. Furthermore, labeled cells could not be detected in the liver by the fluorescent dye or by PCR for the Y-chromosome (Sry-2 gene). In conclusion, optimum conditions to label human hepatocytes with SPIOs were established and did not affect cell viability or metabolic function and were sufficient for in vitro MRI detection. However, the clearance of hepatocytes after transplantation limits the value of MRI for assessing long-term hepatocyte engraftment.

Allogenic hepatocyte transplantation in immunomodulated Lewis rats with acute liver insufficiency following hepatectomy.

Orthotropic Liver transplantation (OLT) is a conventional management for end-stage acute or chronic liver insufficiency, but the shortage of donor organs continues to be the restrictive factor throughout the world. Hepatocyte transplantation (HCTx) might be the promising treatment for several liver diseases and can be used as a "bridge" to OLT. Hepatocytes transplantation can protect and even save human lives, its' applicability remains limited by the large deficiency of liver organs and hepatocytes (HC), and cellular loss after engraftment. Host elimination of grafted cells is called Early Graft Dysfunction. This study was developed for an efficient protocol of HCT. Several conditions have been met in order to achieve a high yield of harvested viable HC, overcome the detached-cell apoptosis, attenuation of innate immune reaction against transplanted cells and a receptive cell environment. HC were isolated from Lewis rats (n = 8) weighing 250 gm, by the 2 step collagen a seper fusion technique, and bone marrow cells (BMCs) were obtained from the rats tibia and femur by centrifugation in a buffer solution. The mean viability of harvested HC and BMCs were 90% and 95% respectively. To minimize the rejection of HC, Lewis rat recipients (n = 14) weighing 250 gm, were irradiated with 6 Gy and received 0.1 mg of anti-aisle GMI antiserum intravenously as immunosuppressive drug. The isolated HC were intra-splenically transplanted and 10(7) bone marrow cells were injected in a penile vein into the recipients on the third day. Simultaneously, 70% hepatectomy and ligation of common bile duct were done. Thirty days later; the grafted spleen had areas with external appearance of a normal liver in ten out of 14 surviving rats (71%). Hematoxlin and eosin (H & E) staining of sections from these fragments showed sinusoids and portal areas, an evidence of successful hepatocyte engraftment and bile canaliculea formation. Large number of HC clusters of 15 to 20 cells and 2 to 4 distended small bile canaliculea were seen per 50 HC. The intrasplenic route for transplanting freshly isolated HC in an immune-compromised animal model was found to give good results regarding cell engraftment and tissue formation.

Human fetal liver cells for regulated ex vivo erythropoietin gene therapy

Possible risks and lack of donor livers limit application of liver transplantation. Liver cell transplantation is, at this moment, not a feasible alternative because engraftment in the liver is poor. Furthermore, there is also shortage of cells suitable for transplantation. Fetal liver cells are able to proliferate in cell culture and could therefore present an alternative source of cells for transplantation. In this study, we investigated the utility of human fetal liver cells for therapeutic protein delivery. We transplanted human fetal liver cells in immunodeficient mice but were not able to detect engraftment of human hepatocytes. In contrast, transplantation of human adult hepatocytes led to detectable engraftment of hepatocytes in murine liver. Transplantation of fetal liver cells did lead to abundant reconstitution of murine liver with human endothelium, indicating that endothelial cells are the most promising cell type for ex vivo liver cell gene therapy. Human liver endothelial cells were subsequently transduced with a lentiviral autoregulatory erythropoietin expression vector. After transplantation in immunodeficient mice, these cells mediated long-term regulation of murine hematocrits. Our study shows the potential of human liver endothelial cells for long-term regulated gene therapy.

Chimeric Mouse Model for the Infection of Hepatitis B and C Viruses

While the chimpanzee remains the only animal that closely models human hepatitis C virus (HCV) infection, transgenic and immunodeficient mice in which human liver can be engrafted serve as a partial solution to the need for a small animal model for HCV infection. The established system that was based on mice carrying a transgene for urokinase-type plasminogen activator (uPA) gene under the control of the human albumin promoter has proved to be useful for studies of virus infectivity and for testing antiviral drug agents. However, the current Alb-uPA transgenic model with a humanized liver has practical limitations due to the inability to maintain non-engrafted mice as dizygotes for the transgene, poor engraftment of hemizygotes, high neonatal and experimental death rates of dizygous mice and a very short time window for hepatocyte engraftment. To improve the model, we crossed transgenic mice carrying the uPA gene driven by the major urinary protein promoter onto a SCID/Beige background (MUP-uPA SCID/Bg). These transgenic mice are healthy relative to Alb-uPA mice and provide a long window from about age 4 to 12 months for engraftment with human hepatocytes and infection with hepatitis C or hepatitis B (HBV) viruses. We have demonstrated engraftment of human hepatocytes by immunohistochemistry staining for human albumin (30-80% engraftment) and observed a correlation between the number of human hepatocytes inoculated and the level of the concentration of human albumin in the serum. We have shown that these mice support the replication of both HBV and all six major HCV genotypes. Using HBV and HCV inocula that had been previously tittered in chimpanzees, we showed that the mice had approximately the same sensitivity for infection as chimpanzees. These mice should be useful for isolating non-cell culture adapted viruses as well as testing of antiviral drugs, antibody neutralization studies and examination of phenotypic changes in viral mutants.

Human Liver Endothelial Cells, But Not Macrovascular or Microvascular Endothelial Cells, Engraft in the Mouse Liver

Liver cell transplantation has had limited clinical success so far, partly due to poor engraftment of hepatocytes. Instead of hepatocytes. other cell types, such as endothelial cells, could be used in ex vivo liver gene therapy. The goal of the present study was to compare the grafting and repopulation capacity of human endothelial cells derived from various tissues. Human endothelial cells were isolated from adult and fetal livers using anti-human CD31 antibody-conjugated magnetic beads. Human macrovascular endothelial cells were obtained from umbilical vein. Human microvascular endothelial cells were isolated from adipose tissue. Cells were characterized using flow cytometry. Liver engraftment and repopulation of endothelial cells was studied after intrasplenic transplantation in monocrotaline-treated immunodeficient mice. Following transplantation, human liver endothelial cells engrafted throughout the mouse liver. With immunoscanning electron microscopy, fenestrae in engrafted human liver endothelial cells were identified, a characteristic feature of liver sinusoidal endothelial cells. In contrast, CD31-negative liver cells, human macrovascular and microvascular endothelial cells were not capable of repopulating mouse liver. Characterization of human liver, macrovascular, and microvascular endothelial cells demonstrated expression of CD31, CD34, and CD146 but not CD45. Our study shows that only human liver endothelial cells, but not macro- and microvascular endothelial cells, have the unique capacity to engraft and repopulate the mouse liver. These results indicate that mature endothelial cells cannot transdifferentiate in vivo and thus do not exhibit phenotypic plasticity. Our results have set a basis for further research to the potential of human liver endothelial cells in liver-directed cell and gene therapy.

Serum Apolipoprotein E as a Marker to Monitor Graft Function After Hepatocyte Transplantation in a Clinically Relevant Mouse Model

IntroductionHepatocyte transplantation, a promising treatment for patients with acute hepatic failure or metabolic liver diseases, requires improvement in engraftment as well as long-term function of the liver cells. We established a hepatocyte transplantation model in apolipoprotein E (ApoE) knockout mice, evaluating serum ApoE and lipoprotein profiles as markers of engraftment of transplanted wild-type hepatocytes. Herein we have described a method to monitor the function of transplanted hepatocytes at low levels of engraftment, corresponding to those reported in clinical cases. We also investigated whether pretreatment with anakinra, an anti-interleukin-1 antagonist, methylprednisolone, or a combination of the two agents improved engraftment. MethodsApoE (−/−) mice were transplanted with hepatocytes isolated from wild-type C57/bl6 mice. A total of 6 × 106 hepatocytes were transplanted by 3 separate intrasplenic injections. Animals were treated before transplantation and daily thereafter for 7 days with anakinra, methylprednisolone, or a combination of both. Graft function was monitored by lipoprotein analysis and quantification of ApoE by enzyme-linked immunosorbent assay. Expression of hepatic ApoE mRNA was quantitated by reverse-transcriptase polymerase chain reaction. ResultsTreatment with anakinra with or without methylprednisolone did not significantly increase serum or hepatic mRNA ApoE expression. The low level of hepatocyte engraftment did not normalize lipoprotein profiles, but produced a significant decline in very low-density lipoprotein and total cholesterol. Repeated transplantations significantly enhanced liver repopulation; serum ApoE levels increased with each infusion, correlating well with hepatic mRNA expression. ConclusionsThe model of serum ApoE, a sensitive marker of engraftment and transplanted hepatocyte function, allowed us to study hepatocyte transplantation in a clinically relevant manner, that is, without pretreatments such as retrorsine or carbon tetrachloride.

In utero transplanted human hepatocytes allow postnatal engraftment of human hepatocytes in pigs

In utero cell transplantation (IUCT) can lead to the postnatal engraftment of human cells in the xenogeneic recipient. Most reports of IUCT have involved hematopoietic stem cells. It is unknown whether human hepatocytes used for IUCT in fetal pigs will lead to the engraftment of these same cells in the postnatal environment. In this study, fetal pigs received direct liver injections of 1 × 10(7) human hepatocytes in utero and were delivered by cesarean section at term. The piglets received a second direct liver injection of 5 × 10(7) human hepatocytes 1 week after birth. The serum was analyzed for human albumin 2, 4, and 6 weeks after engraftment. Piglet livers were harvested 6 weeks after transplantation and were examined by immunohistochemistry, polymerase chain reaction, and fluorescence in situ hybridization for human-specific sequences. Piglets undergoing IUCT with human hepatocytes that were postnatally engrafted with human hepatocytes showed significant levels of human albumin production in their serum at all postengraftment time points. Human albumin gene expression, the presence of human hepatocytes, and the presence of human beta-2 microglobulin were all confirmed 6 weeks after engraftment. IUCT in fetal pigs with human hepatocytes early in gestation allowed the engraftment of human hepatocytes, which remained viable and functional for weeks after transplantation. IUCT followed by postnatal engraftment may provide a future means for large-scale expansion of human hepatocytes in genetically engineered pigs.